The ability to determine the sequence of a polynucleotide is of great scientific importance, as shown by the Human Genome Project in mapping the three billion bases of DNA encoded in the human genome.
The principle method in general use for large-scale DNA sequencing is the chain termination method. This method was first developed by Sanger and Coulson (Sanger et al., Proc. Natl. Acad. Sci. USA, 1977; 74: 5463-5467), and relies on the use of dideoxy derivatives of the four nucleoside triphosphates which are incorporated into the nascent polynucleotide chain in a polymerase reaction. Upon incorporation, the dideoxy derivatives terminate the polymerase reaction and the products are then separated by gel electrophoresis and analysed to reveal the position at which the particular dideoxy derivative was incorporated into the chain.
Although this method is widely used and produces reliable results, it is recognised that it is slow, labour-intensive and expensive.
Fluorescent labels have been used to identify nucleotide incorporation onto a growing nascent DNA molecule, using the polymerase reaction (see WO91/06678). However, these techniques have the disadvantage of increasing background interference from the fluorophores. As the DNA molecule grows, the background “noise” increases and the time required to detect each nucleotide incorporation needs to be increased. This severely restricts the use of the method for sequencing large polynucleotides. The most serious limitation of polynucleotide sequencing systems built around fluorescent dyes, however, is the problem of photobleaching.
Photobleaching is a well documented phenomenon in fluorescent dye systems and results from exposure of the dye to excitation wavelengths. All dye systems have an ability to absorb a limited number of photons before photobleaching occurs. Once photobleaching has occurred the fluorescent dye is no longer visible to the observer and hence, if conjugated to a molecule, this will not be detectable.
There is therefore a need for an improved method for determining the sequence of a polynucleotide, which significantly increases the rate and fragment size of the polynucleotide being sequenced and which preferably does not depend on fluorescently labelled nucleotides for detection. Further, the method should be capable of being carried out by an automated process, reducing the complexity and cost associated with existing methods.